PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB

Abstract

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.histone | mass spectrometry T he NF-κB family is comprised of two protein subfamilies: c-Rel, RelB, and RelA (p65), which include transactivation domains in their C termini, and p100 (p52) and p105 (p50), which include a number of ankyrin repeats in their C termini and have transrepressive functions. All family members include an N-terminal DNA-binding region, the Rel homology domain (1). In the absence of an activating stimulus, NF-κB is located in the cytoplasm in a complex with the inhibitory IκB protein. In the classic pathway of NF-κB activation, extracellular signals activate IκB kinase, which phosphorylates IκBα, leading to its ubiquitination and degradation by proteasomes. NF-κB, liberated from IκB, translocates to the nucleus to regulate its target genes (2). The NF-κB pathway is regulated by means of several different posttranslational modifications, including ubiquitination, phosphorylation, acetylation, sumoylation, and nitrosylation (3). The recent work of several laboratories has revealed the reversible methylation of nonhistone proteins by enzymes that were discovered on the basis of their activities toward histones. For NF-κB, we found that in response to an activating signal, such as treatment with IL-1β, the p65 subunit is reversibly methylated on two specific lysine residues by chromatin remodeling enzymes in ways that profoundly aff...

Histone Arginine Methylation by PRMT7 Controls Germinal Center Formation via Regulating Bcl6 Transcription

“…The similar phenomenon, which is that the expanded myeloid cells and T cells are induced in reaction to autoimmunity and inflammation, was found in mice lacking genes involved in NF-kB activation (45)(46)(47). Previously, NF-kB was also reported to be regulated by other PRMTs (48)(49)(50), suggesting that arginine methylation plays unique roles in NF-kB activation. In addition, it has been reported that histone arginine deimination, which antagonizes arginine methylation, is a response to a wide range of inflammatory stimuli (51,52).…”

Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

“…We demonstrate that PRMT1 forms a cellular complex with RelA and asymmetrically dimethylates the conserved R30 residue in the RelA DNA-binding L1 loop. The asymmetric dimethylation of RelA by PRMT1 is enhanced after prolonged treatment of cells with TNFα and, in contrast to the symmetric modification (16,17), negatively regulates the DNA binding and transcriptional activity of RelA. We also find that down-regulation of PRMT1 enhances the expression of NF-κB target genes after TNFα stimulation by facilitating RelA recruitment to specific promoters.…”

“…The RelA L1 loop, which is critical for nucleotide sequence recognition and binding, has been shown to be monomethylated at K37 by SET7/9 (12) and symmetrically dimethylated at R30 by PRMT5 (16). These modifications cause an increase in the RelA affinity to DNA and activate NF-κB, in contrast with the inhibitory asymmetric R30 dimethylation by PRMT1 identified in our study.…”

“…Several histone lysine methyltransferases, including SET7/9, SETD6, and NSD1, have been shown to both positively and negatively regulate NF-κB through direct monomethylation or dimethylation of distinct lysine residues in RelA (12)(13)(14)(15). Recent studies also have shown that RelA is methylated by the type II protein arginine methyltransferase 5 (PRMT5), which catalyzes the synthesis of ω-N Gmonomethylarginine (MMA) and symmetric ω-N G ,N′ G -dimethylarginine (SDMA) (16,17).…”

Nuclear carbonic anhydrase 6B associates with PRMT5 to epigenetically promote IL-12 expression in innate response

“…Increased activity of the PRMT5-MEP50 complex is found in many kinds of cancer cells and highly correlated with poor prognosis of human cancers, and it is the potential target for treatment of cancers (19). PRMT5 participates in inflammatory response by methylation modification of R35 or R30 of p65 to regulate interleukin-1α (IL-1α), TNF receptor-associated factor 1 (TRAF1), or CXCL10 expression (29). H3R8me2s and H4R3me2s repress gene expression, although the mechanisms mediated with PRMT5 remain to be elucidated (30).…”